The overall goal of this project is to identify specific inhibitors and substrates for the human beta, gamma, and sigma alcohol dehydrogenase (ADH) isoenzymes. Dietary and biogenic alcohols and aldehydes are primarily metabolized by the NAD(H) dependent alcohol dehydrogenase isoenzymes within the cell. There has been considerable debate concerning the involvement of alcohol dehydrogenase isoenzymes in the metabolism of ethanol. The primary reason for this controversy is the lack of specific inhibitors or substrates with which to evaluate their activities in vivo or in vitro. We propose to utilize a structure-based approach toward the identification of specific inhibitors and substrates for the human beta, gamma, and sigma ADH isoenzymes. We have recently solved the three-dimensional structures of all three of these ADH isoenzymes. We have finished the structure refinement of the human beta forms and are continuing the refinement of the gamma and sigma ADH structures. We propose to use these structures to identify inhibitors based on their active site characteristics with the recently developed computer programs DOCK and AUTODOCK. The three- dimensional structures of these isoenzymes will be used in docking experiments to identify specific substrates and inhibitors for these important ADH isoenzymes. Our initial studies will characterize the determinants of specific binding to the respective isoenzymes using a variety of in vitro methods, such as steady-state and stopped-flow kinetic measurements, as well as X-ray crystallography of enzyme-inhibitor complexes. A long term goal of this proposal is to develop stably transformed cell lines expressing one or more of these isoenzymes for evaluation of in vivo inhibitors and toxicological properties of potential inhibitors and substrates. These studies should contribute to the basic understanding of the involvement of these human ADH isoenzymes in the overall rate of ethanol metabolism and may identify important intracellular substrates for these ADH isoenzymes.